Differentially Expressed Proteins in the Liver of HBxTg/p53KO Double Mutant Mouse Using Proteomics
Eun-Sook Park
Department of Medical Science
The Graduate School, Yonsei University
Differentially Expressed Proteins in the Liver of HBxTg/p53KO Double Mutant Mouse Using Proteomics
Directed by Professor Yong-Ho Ahn
The Master's Thesis Submitted to the Department of Medical Science the Graduate School of Yonsei University in Partial Fulfillment of the Requirements for the Degree of Master of Medical Science
Eun-Sook Park
December 2003
This certifies that the master's thesis
of Eun-Sook Park is approved.
------Thesis Supervisor : Yong-Ho Ahn
------Jeon-Han Park : Thesis Committee Member#1
------Je-Kyung Seong : Thesis Committee Member#2
The Graduate School
Yonsei University
December 2003 Contents
Abstracts··························································································································· 1
I. Introduction··············································································································· 4
II. Materials and Methods ··················································································· 7
1. Laboratory animals························································································ 7
2. Establishment of HBxTg/p53KO double mutant mice································ 7
A. Hybrid between HBxTg and p53KO mice ············································ 7
B. Genotyping of HBxTg/p53KO double mutant mice····························· 8
(A) DNA extraction················································································· 8
(B) PCR ··································································································· 9
3. Liver function test························································································· 13
4. Histology of liver··························································································· 13
5. Proteomic Analysis ······················································································· 13
A. Sample preparation··············································································· 13
B. 2D-gel electrophoresis ········································································· 14 C. Protein image analysis··········································································· 15
D. Identification by MALDI-TOF MS ··················································· 15
III. Results ········································································································ 16
1. Liver function test·························································································· 16
2. Histology of liver ·························································································· 19
3. Proteomic analysis of liver ············································································ 21
A. Identification of HBx overexpression specifically expressed proteins in
the liver by 2-DE and MALDI-TOF MS ············································ 21
B. Identification of specifically expressed proteins related with the
functional loss of p53 by 2-DE and MALDI-TOF MS························· 35
C. Identification of specifically expressed proteins in the liver
HBxTg/p53KO double mutant mice by 2-DE and MALDI-TOF MS···· 45
D. Summary of specifically expressed proteins profiles among groups ······ 53
IV. Discussion ············································································································ 61
V. Co n c l us io n ··············································································································· 63 Reference ························································································································ 65
Abstracts (in Korea)······························································································· 70
List of Figures
Figure 1. Establishment of HBxTg/p53KO double mutant mice
Figure 2. Genotyping of HBxTg/p53KO double mutant mice
Figure 3. The changes of liver weight in B6, HBxTg, p53KO and HBxTg/p53KO
double mutant mice
Figure 4. Serum value of alanine aminotransferase in B6, HBxTg, p53KO and
HBxTg/p53KO double mutant mice
Figure 5. Histopathological findings of liver by hematoxylin and eosin staining
Figure 6. 2-DE images of master gel in the liver of B6, HBxTg, p53KO and
HBxTg/p53KO double mutant mice visualized by Coomassie blue
staining
Figure 7. 2-DE images of differentially expressed proteins in the liver of B6,
HBxTg, p53KO and HBxTg/p53KO double mutant mice
List of Tables
Table 1. Sequence of PCR primers
Table 2. Lists of up- and down-regulated proteins in the liver of HBxTg mice
compared to B6 mice Table 3. Differentially expressed protein function of the liver in HBxTg mice
compared to B6 mice
Table 4. Lists of up- and down-regulated proteins in the liver of p53KO mice
compared to B6 mice
Table 5. Differentially expressed protein function of the liver in p53KO mice
compared to B6 mice
Table 6. Lists of up- and down-regulated proteins in the liver of HBxTg/p53KO
double mutant mice compared to B6 mice
Table 7. Differentially expressed protein function of the liver in HBxTg/p53KO
double mutant mice compared to B6 mice
Table 8. Differentially expressed protein profiles of the liver in HBxTg, p53KO
and HBxTg/p53KO double mutant mice compared to B6 mice
Abbreviations
B6 mice : C57BL/6J mice
HBxTg mice : C57BL/6J-HBx/HBx transgenic mice
p53KO mice : C57BL/6J- p53-/ p53- knock-out mice
CHAPS : 3-[(3-cholamidopropyl)dimthylammonio]-1-propanesulfonic acid
DTT : dithiothreitol
2DE : two-dimensional gel eletrophoresis
IEF : isoelectrofocusing
MALDI-TOF MS : matrix-assisted laser desorption ionization time of flight mass spectrometry
IPG : immobilized pH gradient Abstract
Differentially Expressed Proteins in the Liver of
HBxTg/p53KO Double Mutant Mouse Using Proteomics
Eun-Sook Park
Department of Medical Science
The Graduate School, Yonsei University
(Directed by Professor Yong-Ho Ahn )
Hepatitis B virus (HBV) has been clearly recognized as an etiological factor for hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein. However, little was known for the mechanism underlying HBx-mediated oncogenecity. Functional inactivation of p53 by complex formation with HBx has been regarded as one of possible mechanisms of HCC in HBV-infected liver.
Although there have been many attempts to investigate the role of p53 which was one of well-characterized tumor suppressive proteins, in vivo relation between p53 and
1 HBx is not fully understood during hepatocellular carcinogenesis caused by HBx.
Therefore, we aimed to elucidate the molecular mechanism of HCC and to discover the effect of functional loss of p53 on the development of HCC by HBx. We generated HBx transgenic and p53 knock-out double mutant mice (HBxTg/p53KO) by the cross mating of HBx transgenic mice (HBxTg) to p53 knock-out (p53KO) mice. Serum ALT value was determined and liver section was observed under H&E staining. We employed two dimensional polyacrylamide gel electrophoresis (2DE) and matrix assisted laser desorption/ionization time of flight mass spectrometry
(MALDI-TOF MS) to determine profiles of differentially expressed proteins in the liver of 1-month-old male HBxTg, p53KO and HBxTg/p53KO mice compared to their age-matched normal mice (B6). In HBxTg/p53KO mice, ALT value were significantly increased compared to normal B6 mice and even, both HBxTg and p53KO mice. HBx/p53KO double mutant mice showed swelling of hepatocytes and irregular hepatic cord histopathologically. However, there is not distinct change in the liver of B6, HBxTg and p53KO mice. Cell cycle, cytoskeleton, several metabolisms related proteins were mainly differentially expressed in the liver of HBxTg mice compared to B6 mice. Signal transduction pathway, ATP binding and chaperone activity and lipid binding related proteins were differentially expressed in the liver of p53KO mice compared to B6 mice. Cell cycle, several metabolisms, ATP binding and chaperone activity, transferase activity related proteins were differentially expressed in the liver of HBxTg/p53KO double mutant mice compared to B6 mice. We identified 24 liver proteins, which were differentially regulated in each of HBxTg,
2 p53KO and HBxTg/p53KO mice compared with B6 mice. They are proteins related with cell cycle regulation, lipid metabolism, fatty acid metabolism, ATP synthesis and several metabolism related proteins. The proteins identified provide insights into the
HCC caused by HBx and the regulation of this process related with the functional loss of p53.
------
Key words: hepatocellular carcinoma (HCC), HBx, p53, HBxTg/p53KO double
mutant mice, liver.
3 Differentially Expressed Proteins in the Liver of
HBxTg/p53KO Double Mutant Mouse Using Proteomics
Eun-Sook Park
Department of Medical Science
The Graduate School, Yonsei University
(Directed by Professor Yong-Ho Ahn )
I. Introduction
Hepatocellular carcinoma (HCC) is known as a common malignant tumor that takes the lives of about one million people world wide annually. The major risk factors for human HCC are Hepatitis B virus (HBV), Hepatitis C virus (HCV), p53 mutation, aflatoxin, alcohol abuse and male gender.1,2 In addition, development of
HCC is linked to cirrhosis.3 Epidemiological data highly indicated that there is a consistent and specific causal association between HBV infection and occurrence of
HCC.4,5 HCC is on the increase in many countries where HCV infection is more
4 prevalent than HBV infection.5,6 In Korea, however, there are much more HBV infections than HCV infections. The hepatitis B virus (HBV) genome contains four transcriptional open reading frames designated S, C, P, and X 7,8 . Of the four viral- encoded proteins, Hepatitis B virus X (HBx) has been identified as potentially oncogenic. 9 Since the oncogenic role of HBx in a transgenic mouse model was first demonstrated, there have been controversial results by several researchers. In some studies, HBx induced no pathology in the livers of HBx transgenic mice (HBxTg), but it sensitized liver cells to the other carcinogenic effects.10,11,12,13 But in other studies, transgenic expression of the HBx was shown to induce liver tumors in transgenic mice.9,14 HBx protein is a transactivator without DNA-binding activity. 15,16 HBx can also bind to the p53 tumor suppressor protein and interfere with the role of p53 which plays in the cellular response to DNA damage.17 Initial investigations on the interaction between HBx and p53 demonstrated that the two proteins associate with each other both in vivo and in vitro.18 Inactivation of p53 by complex formation with
HBx has been suggested as a possible mechanism of HCC. 17,19-22 Contrary to the previous reports, HBx has been reported to exert a spontaneous proapoptotic effect on primary cultures of hepatocytes and in the livers of HBxTg mice.23-25 These observations in vivo and in vitro suggested that HBx regulates endogenous cellular pathways. However, still the effect of p53 on the HCC caused by HBx remains unclear. More recently, HBxTg/p53KO (HBx/HBx // p53-/p53-) double mutant mice were generated by the cross-mating of HBxTg mice with p53 knock-out mice
5 (p53KO) 26, which is a very useful animal model for studying the relations and the role of p53 on the development of HCC caused by HBx.
To elucidate factors involved in HCC caused by HBx under the functional loss of p53, we employed two dimensional polyacrylamide gel electrophoresis (2DE) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-
TOF MS) and determined profiles of differentially expressed proteins in the liver of
1-month-old male HBxTg, p53KO and HBxTg/p53KO mice compared to its age- matched normal mice (B6). Also serum ALT value was determined and liver section was observed under H&E staining.
6 II. Materials and methods
1. Laboratory Animals
We used HBxTg mice (C57BL/6J-HBx/HBx transgenic mice) developed by Dr Yu.
14 p53KO mice (C57BL/6J- p53-/ p53- knock-out mice) were purchased from the
Jackson Laboratory (U.S.A.). 26 HBxTg/p53KO double mutant mice (HBx/HBx//p53-
/p53-) were established by the cross mating of HBxTg mice to p53KO mice. Age matched wild type mice were used as controls from the same background of HBxTg mice. All animals have been maintained in SPF barrier area of the Department of
Laboratory Animal Medicine, Yonsei University College of Medicine. The temperature (22±2℃), humidity (50±5%) and light (12D/12N) of animal rooms were controlled constantly. The chloride-added (RO water) water was supplied ad libitum. All animals were sacrificed at the age of 1-month old. Liver was removed, weighted and stored at -70℃ until used.
2. Establishment of HBxTg/p53KO double mutant mice
A. Hybrid between HBxTg and p53KO mice
7
HBxTg mice were mated with p53KO mice to generate F1 hybrid (HBx/+//p53-/+).
F1 pairs were intercrossed each other to generate F2 hybrid when they were 8 weeks old (Fig. 1). We selected HBxTg/p53KO (HBx/HBx//p53-/p53-) double mutant mice from F2 hybrids by genotyping.
B. Genotyping of HBxTg/p53KO double mutant mice
(A) DNA extraction
DNA was isolated from tail as follows. Tail was cut with a sterile razor blade and solubilized in 1 ㎖ lysis buffer (1% SDS, 5 mM EDTA, 10 mM Tris-HCl pH 7.6,
100 mM NaCl, 200 ㎍/㎖ proteinase K). The buffer was incubated in dry oven at
60℃ overnight. After incubation, 400 ㎕ solution (4.21 M NaCl, 0.63 M KCl, 10 mM Tris-HCl) was added with gentle inversion for protein precipitation. The mixture was centrifuged at 13,000 rpm for 30 minutes at 4℃. 300 ㎕ upper aqueous phase containing DNA and 300 ㎕ 100% ethanol were mixed in a microcentrifuge and were incubated in a refrigerator for 2 hours at -20℃. The mixture was centrifuged at
13,000 rpm for 20 minutes at 4℃ and the DNA pellet was washed three times with cold 70% ethanol. The DNA pellet was dried and resuspended in double-distilled water. Purity and concentration of the final samples were evaluated by ultraviolet
8 spectrophotometer.
(B) PCR
Primer sets for HBx gene and for discriminating HBx homozygote from heterozygote, were obtained from Dr Yu (KRIBB, Taejon, Korea).14 Primer sets for p53 gene was provided by commercial service.26 Twenty ㎕ reaction solution containing 2 ㎍ of the genomic DNA was used for PCR. The primer sequences, annealing temperature and PCR cycles are described in Table 1. After electrophoresis, male mice with genotype of HBxTg/p53KO double mutant mice (HBx/HBx //p53-
/p53-) were discriminated and sacrificed (Fig. 2).
9
Table 1. Sequence of PCR primers
Genotype Primer Sequence Tm Cycles p1 5'-CTTGGGTGGAGAGGCTATTC-3'
p2 5'-AGGTGAGATGACAGGAGATC-3' 64℃ 12 +/+, +/p53- (-0.5) + or p53-/p53- p3 5'-ATAGGTCGGCGGTTCAT-3' 58℃ 25
p4 5'-CCCGAGTATCTGGAAGACAG-3
p1 5'-TTCTCATCTGCCGGTCCGTG-3' HBx/? or +/+ 53℃ 34 p2 5'-CTCTCTTTGCGCTCCCTGGG-3'
p1 5'-GAAAACACACTCACTGTTCAGAG-3' HBx/HBx 54℃ 34 or HBx/+ p2 5'-GGGTCAATGTCCATGCCCCA-3'
10
HBxTg mice p53KO mice
(HBx/HBx )X (p53-/p53-)
F1 (HBx/+ // p53-/+) X (HBx/+ // p53-/+)
HBx p53 1 HBx/HBx + / + 2 HBx/HBx + / - 3 HBx/HBx - / - 4 HBx/+ + / + F2 5 HBx/+ + / - 6 HBx/+ - / - 7 +/+ + / + 8 +/+ + / - 9 +/+ - / -
Figure 1. Establishment of HBxTg/p53KO double mutant mice. The homozygotic
HBxTg mice (HBx/HBx) were mated with the homozygotic p53KO mice (p53-/p53-) to generate F1 hybrid (HBx/+//p53-/+). F1 hybrid pairs were intercrossed each other to generate F2 hybrid. We selected HBxTg/p53KO (HBx/HBx//p53-/p53-) double mutant mice from F2 hybrids.
11
A B
M 1 2 3 4 5 6 M 1 2 3 4 p53 wild HBx/HBx (homozygote) HBx HBx /+ p53KO (heterozygote)
Figure 2. Genotyping of HBxTg/p53KO double mutant mice. (A) PCR genotyping
of HBx transgene and p53 knock-out. M; DNA size marker. Lane 1,2 and 3 showed
the introduction of HBx transgene. Lane 4,5 and 6 showed wild type of HBx. Lane 4
showed the heterozygotic knock-out of p53. Lane 5 showed the homozygotic knock-
out of p53. Lane 6 showed wild type p53 (B) PCR genotyping for heterozygote and
homozygote of HBx transgene. M; DNA size marker. Lane 1 and 2 showed the
homozygotic transgene of HBx. Lane 3 and 4 showed the heterozygotic transgene of
HBx.
12 3. Liver Function Test
Under anesthesia with ethyl-ether, blood was collected by heart puncture from mice.
Serum level of alanine aminotransferase (ALT) was determined by commercial service provided by Ewon Reference Laboratory, Seoul, Korea.
4. Histology of liver
Liver samples were fixed in 10% neutral buffered formalin for 24 hours. After serial processing of dehydration by graded alcohol series and clearing, tissues were embedded in paraffin. Tissues were cut into 4 ㎛ sections. Slides were stained with hematoxylin & eosin.
5. Proteomic analysis
A. Sample preparation
Liver tissue of 100 ㎎ was homogenized under liquid nitrogen. Homogenized liver tissue was lysed with 800 ㎕ lysis buffer (7 M urea, 2 M thiourea, 2% w/v CHAPS,
2% Pharmalyte pH 3-10, 100 mM DTE) and centrifuged at 50000 rpm at 4℃ for 1 hr.
13 The supernatant was transferred into new eppendorf tube. After then the supernatant mixed DNase (Roche 104132, 2.5 ㎎/㎖) and Rnase (Roche 019141, 2.5 ㎎/㎕) and incubated for 30 min on 20℃. Protein concentrations were determined by the
Bradford protein assay (Bio-Rad, Switzerland). Solubilized protein samples were divided into 1 mg aliquots and stored at -80℃.
B. 2D-gel electrophoresis
2D-PAGE was performed as previously described. 27 The 1 mg aliquots proteins were diluted in lysis buffer to total volume 450 ㎕. The sample was applied to the
240 mm, immobilized, nonlinear pH gradient strips of pH 3-10 (IPG Drystrips,
Amersham Pharmacia Biotech. Uppsala, Sweden), which was rehydrated for at least
10 hrs. After rehydration, the strips were focused at 30 V for 3 hrs, 100 V for 1 hr, 200
V for 1 hr, 500 V for 1 hr, 1,000 V for 1 hr and finally at 8,000 V for 11 hrs so as to obtain approximately 90,000 Vhr (IPG phore, Amersham Pharmacia Biotech). Once
IEF was completed, the strips were equilibrated in 6M urea containing 20% v/v glycerol, 2% w/v SDS and 0.01% w/v BPB with 10mM TBP (Tributyl phosphine,
Fluka Chemie, Buchs, Switzerland). SDS-PAGE was performed using 8-18% SDS polyacrylamide separating gel without a stacking gel using the Ettan Dalt system
(Amersham Pharmacia). The second dimensional was carried out overnight at 3W/gel at 20℃. The gels were stained with Coomassie G-250 (Bio-Rad, USA).
14 C. Protein image analysis
The stained gels were scanned using GS800 photometer (Bio-Rad, U S A) and analyzed with the Melanie III (SIB, Swiss). For comparison of spot density, the digitized image was compared by matching method. Intensity levels were normalized between gels as a proportion of the total protein intensity detected for the entire gel.
Differentially expressed spots among groups by > 3-fold and < 1/3-fold were analyzed and annotated.
D. Protein identification by MALDI-TOF MS
MALDI-TOF MS was performed using the Applied Biosystems Voyager DE-PRO spectrometer (Applied Biosystems, Forste city, USA), equipped with a 337 nm nitrogen laser. The instrument was operated in accelerating voltage 20 kV, positive ion reflection mode, voltage grid 74.5 %, guide wire voltage 0 %, delay-time 120 ns.
The spectra were internally calibrated using the trypsin autolysis products (842.51
[M+H] and 2211.11 [M+H]), and the proteins were identified by searching in Swiss-
Prot and NCBI database using MS-FIT (Protein Prospector, UCSF, San Francisco, CA,
USA). All the searches were analyzed with a 50 ppm mass tolerance. For the detection of post-translational modifications, the molecular weight of each spots were analyzed on 3 different gels with MALDI-TOF.28
15 II. Results
1. Liver weight and liver function test
The liver weight in HBxTg, p53KO and HBxTg/p53KO double mutant mice were significantly increased compared to B6 mice (p<0.05, Fig. 3). Serum ALT level of
HBxTg/p53KO double mutant mice was significantly higher than that those of B6,
HBxTg and p53KO mice (p<0.05, Fig. 4).
16
(g) * 1.4 * 1.2 *
1.0 (5) (5) (11) 0.8 (5) 0.6 0.4
0.2
0.0
Figure 3. The changes of liver weight in B6, HBxTg, p53KO and HBxTg/p53KO double mutant mice. Data were expressed as means±S.D. The liver weight in
HBxTg, p53KO and HBxTg/p53KO double mutant mice were significantly increased compared to B6 mice (*: p<0.05). However, there is not significant difference in the liver weight among HBxTg, p53KO and HBxTg/p53KO double mutant mice.
Number of parentheses indicated the number of animals used. ( B6 mice, HBxTg mice, p53KO mice, HBxTg/p53KO double mutant mice)
17
ALT (IU/l)
250.0 (11) 200.0 *
150.0
100.0
50.0 (5) (5) (5) 0.0
Figure 4. Serum value of alanine aminotransferase in B6, HBxTg, p53KO and
HBxTg/p53KO double mutant mice. Data were expressed as means±S.D. The ALT value was significantly increased in HBxTg/p53KO double mutant mice compared to
B6, HBxTg and p53KO mice (*: p<0.05). ( B6 mice, HBxTg mice, p53KO mice, HBxTg/p53KO double mutant mice)
18 2. Histology of liver
We observed liver stained with by hematoxylin & eosin. Consequently, in 1 month of age, the livers of HBxTg mice were characterized by a generalized variation in the nuclear size of the hepatocytes, compared to B6 mice (Fig. 5A and B). Around the central vein there were hepatocytes with large and light nuclei, while hepatocytes with small and dark nuclei were observed around the portal vein in HBxTg mice (Fig.
5B). In 1-month-old HBxTg/p53KO double mutant mice, the livers were characterized by a generalized variation in the size of the hepatocytes and their nuclei
(Fig. 5D). Around the central vein there were hepatocytes with large nuclei showing chromatin clumping, while hepatocytes with small nuclei were observed around the portal vein (Fig. 5D).
19
A B
C D
Figure 5. Histopathological findings of liver by hematoxylin and eosin staining.
HBx/p53KO double mutant mice showed swelling of hepatocytes and irregular hepatic cord histopathologically. However, there is not distinct change in the liver of
B6 HBxTg and p53KO mice.
A. Liver of 1-month-old B6 mouse
B. Liver of 1-month-old HBxTg mouse
C. Liver of 1-month-old p53KO mouse
D. Liver of 1-month-old HBxTg/p53KO double mutant mouse
20 3. Proteomic analysis of liver
A. Identification of HBx overexpression specifically expressed proteins in the liver by 2-DE and MALDI-TOF MS
We identified profiles of differentially expressed liver proteins in HBxTg, p53KO and HBxTg/p53KO double mutant mice compared to normal B6 mice. Liver proteins were extracted and analyzed by 2D-PAGE. After 2D gels were visualized by
Coomassie blue staining, 2-DE was executed three times for each type of tissue sample from each mouse (Fig. 6). Within each pair (e.g., B6 vs. HBxTg, B6 vs. p53KO, B6 vs. HBxTg/p53KO), the percent volume of the protein spots was compared between each pair using Melanie III. The protein spots that changed > 3 fold up-regulation or < 1/3 fold down-regulation were selected. The selected spots were cut out from the gel and subjected to in-gel digestion with trypsin and peptide fingerprinting by MALDI-TOF MS. The peptide mass data were identified by MS-
FIT. In this way, we found up- and down-regulated proteins each pair (Table 2, 4 and
6).
It was 54 proteins changed > 3-fold up-regulation or < 1/3-fold down-regulation in the liver of HBxTg compared to B6 mice (Table 2). 54 proteins were found to be affected by HBx transgene. 54 proteins search for protein function by http://www.ebi.ac.uk/ego/ (Table 3). Among them, oxidoreductase activity
21 (metabolism resulting in cell Growth) related proteins were 9. For example, aldehyde dehydrogenase family 7 member A1, adehyde dehydrogenase mitochondrial precursor, malate dehydrogenase, 2-oxoisovalerate dehydrogenase alpha subnuit, peroxisomal bifunctional enzyme, sepiapterin reductase were up-regulated proteins, dimethylglycine dehydrogenase, NADH-ubiquinone oxidoreductase 75kDa subunit and peroxiredozin 1 were down-regulated proteins. ATP binding and chaperone activity related proteins were 9. For example, 60kDa heat shock protein mitochondrial precursor, seryl-tRNA synthetase, Stress-70 mitochondrial precursor were up- regulated proteins. 26S proteasome non-ATPase regulatory subunit 13, 26S protease regulatory subunit 6B, S-adenosylmethionine synthetase alpha and beta forms, ATP synthase beta chain mitochondrial precursor, glutathione synthetase, methylcrotonyl-
CoA carboxylase alpha chain mitochondrial precursor were down-regulated proteins.
Lipid binding and transport related proteins were 5. For example, apolipoprotein E precursor, nonspecific lipid transfer protein mitochondrial precursor, SEC14-like protein 2 were up-regulated proteins. Microsomal triglyceride transfer protein large subunit and PCTP-like protein (PCTP-L) were down-regulated protein. Hydrolase activity related proteins were 3. For example, proteasome subunit alpha type 4, proteasome subunit alpha type 5, transitional endoplasic reticulum ATPase were up- regulated proteins. Calcium ion binding related proteins were 2. Calreticulin precursor was up-regulated protein. Transketolase was down-regulated protein. Cell cycle regulation and signal transduction related proteins were 3.They were zinc finger protein 189 that be related with DNA replication and ribonuclease/angiogenin
22 inhibitor 1 which is related with ribonuclese inhibitor. These proteins are mainly related with cell cycle regulation. Up-regulated Heterogeneous nuclear ribonucleoproteins A2/B1 and Down-regulated poly (A) polymerase beta were proteins related with RNA processing. Up-regulated guanie nucleotide-binding protein beta subunit-like protein and Down-regulated Pyruvate kinase were proteins related with kinase activity. Down-regulated 10-formyltetrahydrofolate dehydrogenase and thiosulfate sulfurtransferase were proteins related with transferase activity. Down-regulated keratin type I cytoskeletal 18 and keratin type II cytoskeletal
18 were proteins related with cytoskeleton. Remaining 8 were aflatozin biosynthesis, isomerase activity, protein biosynthesis, cell mobility, GTP binding, aminoacylase activity, lyase activity and ligase activity related proteins. Function of 4 proteins could not search and 3 proteins were RIKEN cDNA.
23
pI 3 10 pI 3
A B
pI 3 10 pI 3 10
C D
24 Figure 6. 2-DE images of master gel in the liver of B6, HBxTg, p53KO and
HBxTg/p53KO double mutant mice visualized by Coomassie blue staining.
Proteins from the whole liver were extracted and separated on pH2 to 10 nonlinear immobilized pH-gradient strips, followed by a 8-18% SDS polyacrylmaide gel. The gel was stained with Coomassie blue G-250. A. B6 mice, B. HBxTg mice, C. p53KO mice, D. HBxTg/p53KO double mutant mice.
25 Table 2. Lists of up- and down-regulated proteins in the liver of HBxTg mice compared to B6 mice
B6 vs HBxTg Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Aflatoxin B1 aldehyde reductase 1 27527042 32 37677/6.4 MUS Up MUSCULUS Aldehyde dehydrogenase family 7 Q9DBF1 26 55645/6 MOUSE Up member A1 (Antiquitin 1) Aldehyde dehydrogenase, mitochondrial P47738 23 56538/7.5 MOUSE Up precursor (ALDH class 2) (AHD-M1) (ALDHI) (ALDH-E2) Apolipoprotein E precursor (Apo-E) P08226 39 35867/5.6 MOUSE Up Calreticulin precursor (CRP55) P14211 27 47995/4.3 MOUSE Up (CALREGULIN) (HACBP) (ERP60) gi|13385656|ref|NP_080428.1| RIKEN 13385656 33 34644/7.6 UNREADABLE Up cDNA 0610010D20 [Mus musculus] gi|19527178|ref|NP_598721.1| RIKEN 19527178 18 61941/5.7 UNREADABLE Up cDNA 9130231C15 [Mus musculus] gi|21312204|ref|NP_077219.1| RIKEN 21312204 39 28027/6.3 UNREADABLE Up cDNA 2810435D12 [Mus musculus]
gi|6754472|ref|NP_004847.2| kinesin 6754472 11 98106/8.7 UNREADABLE Up family member 23 isoform 2; mitotic kinesin-like 1; kinesin-like 5 (mitotic kinesin-like protein 1) [Homo sapiens]
Guanine nucleotide-binding protein beta P25388 52 35077/7.6 HUMAN Up subunit-like protein 12.3 (P205) (Receptor of activated protein kinase C 1) (RACK1) (Receptor for activated C kinase)
60 kDa heat shock protein, mitochondrial P19226 36 60956/5.9 MOUSE Up precursor (Hsp60) (60 kDa chaperonin) (CPN60) (Heat shock protein 60) (HSP- 60) (Mitochondrial matrix protein P1) (HSP-65) Heterogeneous nuclear O88569 45 35993/8.7 MOUSE Up ribonucleoproteins A2/B1 (hnRNP A2 / hnRNP B1) Malate dehydrogenase, cytoplasmic P14152 31 36477/6.2 MOUSE Up Nonspecific lipid-transfer protein, P32020 17 59158/7.2 MOUSE Up mitochondrial precursor (NSL-TP) (Sterol carrier protein 2) (SCP-2) (Sterol carrier protein X) (SCP-X) (SCPX)
26 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI 2-oxoisovalerate dehydrogenase alpha P11960 24 50165/7.7 RAT Up subunit, mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha) Peroxisomal bifunctional enzyme (PBE) Q9DBM2 21 78244/9.3 MOUSE Up (PBFE) [Includes: Enoyl-CoA hydratase ; 3,2-trans-enoyl-CoA isomerase ; 3- hydroxyacyl-CoA dehydrogenase ] Proteasome subunit alpha type 4 Q9R1P0 30 29471/7.6 MOUSE Up (Proteasome component C9) (Macropain subunit C9) (Multicatalytic endopeptidase complex subunit C9) (Proteasome subunit L) Proteasome subunit alpha type 5 Q9Z2U1 30 26411/4.7 MOUSE Up (Proteasome zeta chain) (Macropain zeta chain) (Multicatalytic endopeptidase complex zeta chain)
Ribonuclease/angiogenin inhibitor 1 16307569 33 49817/4.7 MUS Up MUSCULUS SEC14-like protein 2 (Alpha-tocopherol Q99J08 32 46301/6.7 MOUSE Up associated protein) (TAP)
Selenium-binding protein 1 (56 KDa P17563 19 52352/6 MOUSE Up selenium-binding protein) (SP56)
Selenium-binding protein 2 (56 kDa Q63836 37 52629/5.8 MOUSE Up acetaminophen-binding protein) (AP56)
Sepiapterin reductase (SPR) Q64105 53 27883/5.6 MOUSE Up
Seryl-tRNA synthetase (Serine--tRNA P26638 38 58389/5.9 MOUSE Up ligase) (SerRS) Stress-70 protein, mitochondrial P38647 21 73529/5.9 MOUSE Up precursor (75 kDa glucose regulated protein) (GRP 75) (Peptide-binding protein 74) (PBP74) (P66 MOT) (Mortalin) Transitional endoplasmic reticulum Q01853 15 89309/5.1 MOUSE Up ATPase (TER ATPase) (15S Mg(2+)- ATPase p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin]
Triosephosphate isomerase (TIM) P17751 27 26713/6.9 MOUSE Up 60S acidic ribosomal protein P0 (L10E) P14869 32 34187/5.9 MOUSE Down Actin-like protein 3 (Actin-related protein P32391 39 47372/5.6 HUMAN Down 3) (Actin-2)
27 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI S-adenosylmethionine synthetase alpha Q00266 28 43648/5.9 HUMAN Down and beta forms (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III)
Alpha enolase (2-phospho-D-glycerate P17182 21 47141/6.4 MOUSE Down hydro-lyase) (Non-neural enolase) (NNE) (Enolase 1) ATP synthase beta chain, mitochondrial P56480 31 56301/5.2 MOUSE Down precursor Dimethylglycine dehydrogenase , Q63342 14 96048/6.9 MOUSE Down mitochondrial precursor (ME2GLYDH)
Elongation factor 2 (EF-2) P05197 8 95285/6.4 RAT Down
10-formyltetrahydrofolate dehydrogenase Q8R0Y6 25 98710/5.6 MOUSE Down (10-FTHFDH) gi|13384746|ref|NP_079647.1| 13384746 53 45795/5.8 UNREADABLE Down aminoacylase 1 [Mus musculus]
gi|31982147|ref|NP_775547.2|hexose-6- 31982147 13 89911/6.6 UNREADABLE Down phosphate dehydrogenase (glucose 1- dehydrogenase) [Mus musculus] Glutathione synthetase (Glutathione P51855 33 52247/5.6 MOUSE Down synthase) (GSH synthetase) (GSH-S)
Keratin, type I cytoskeletal 18 P05784 36 47504/5.2 MOUSE Down (Cytokeratin 18) (Cytokeratin endo B) (Keratin D) Keratin, type II cytoskeletal 8 P11679 17 54450/5.5 MOUSE Down (Cytokeratin 8) (Cytokeratin endo A)
Methylcrotonyl-CoA carboxylase alpha Q99MR8 29 79345/7.7 MOUSE Down chain, mitochondrial precursor (3- Methylcrotonyl-CoA carboxylase 1) (MCCase alpha subunit) (3- methylcrotonyl-CoA:carbon dioxide ligase alpha subunit) Microsomal triglyceride transfer protein, O08601 33 99143/7.8 MOUSE Down large subunit precursor
NADH-ubiquinone oxidoreductase 75 kDa P28331 18 79574/5.8 HUMAN Down subunit, mitochondrial precursor (Complex I-75Kd) (CI-75Kd)
PCTP-like protein (PCTP-L) (StAR- Q9JMD3 31 32952/6.7 MOUSE Down related lipid transfer protein 10) (StARD10) (START domain-containing protein 10) (Serologically defined colon cancer antigen 28 homolog)
28 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Peroxiredoxin 1(Thioredoxin peroxidase P35700 47 22177/8.3 MOUSE Down 2) (Thioredoxin-dependent peroxide reductase 2) (Osteoblast specific factor 3) (OSF-3) (Macrophage 23 kDa stress protein) Poly(A) polymerase beta (PAP beta) Q9NRJ5 18 71682/6.0 MOUSE Down (Polynucleotide adenylyltransferase beta) (Testis-specific poly(A) polymerase)
26S protease regulatory subunit 6B P43686 29 47367/5.1 HUMAN Down (MIP224) (MB67 interacting protein) (TAT-binding protein-7) (TBP-7)
26S proteasome non-ATPase regulatory Q9WVJ2 34 42810/5.5 MOUSE Down subunit 13 (26S proteasome regulatory subunit S11) (26S proteasome regulatory subunit p40.5) Pyruvate kinase, isozymes R/L (L-PK) P53657 22 62309/6.6 MOUSE Down
Serine/threonine protein phosphatase 2A, P30153 36 65224/5.0 HUMAN Down 65 KDA regulatory subunit A, alpha isoform (PP2A, subunit A, PR65-alpha isoform) (PP2A, subunit A, R1-alpha isoform) (Medium tumor antigen- associated 61 KDA protein) Succinyl-CoA ligase[GDP-forming] beta- Q9Z2I8 19 43858/5.9 MOUSE Down chain, mitochondrial precursor(Succinyl- CoA synthetase, betaG chain) (SCS- betaG) (GTP-specific succinyl-CoA synthetase beta subunit) Thiosulfate sulfurtransferase (Rhodanese) P52196 48 33466/7.7 MOUSE Down
Transketolase (TK) (P68) P40142 29 67631/7.2 MOUSE Down
Zinc finger protein 189 O75820 16 72979/8.8 HUMAN Down
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold]
29 Table 3. Functional annotation of differentially expressed proteins in the liver of HBxTg mice compared to B6 mice
Accession Function Protein name Species Regulation # Oxidoreductase Aldehyde dehydrogenase family 7 member A1 Q9DBF1 MOUSE Up activity (Antiquitin 1) (metabolism resulting in cell growth) Aldehyde dehydrogenase, mitochondrial precursor P47738 MOUSE Up (ALDH class 2) (AHD-M1) (ALDHI) (ALDH-E2)
Malate dehydrogenase, cytoplasmic P14152 MOUSE Up (malate degydrogenase activity)
2-oxoisovalerate dehydrogenase alpha subunit, P11960 RAT Up mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha)
Peroxisomal bifunctional enzyme (PBE) (PBFE) Q9DBM2 MOUSE Up [Includes: Enoyl-CoA hydratase ; 3,2-trans-enoyl-CoA isomerase ; 3-hydroxyacyl-CoA dehydrogenase ] (catalytic activity, ion transport : ADP, ATP carrier protein) Sepiapterin reductase (SPR) Q64105 MOUSE Up
Dimethylglycine dehydrogenase , Q63342 MOUSE Down mitochondrial precursor (ME2GLYDH) (electron transport)
NADH-ubiquinone oxidoreductase 75 kDa subunit, P28331 HUMAN Down mitochondrial precursor (Complex I-75Kd) (CI-75Kd) (NADH dehydrogenase activity, electron transport activity)
Peroxiredoxin 1(Thioredoxin peroxidase 2) P35700 MOUSE Down (Thioredoxin-dependent peroxide reductase 2) (Osteoblast specific factor 3) (OSF-3) (Macrophage 23 kDa stress protein) (antioxidant activity, peroxidase activity)
ATP binding and 60 kDa heat shock protein, mitochondrial precursor P19226 MOUSE Up chaperone (Hsp60) (60 kDa chaperonin) (CPN60) (Heat shock activity protein 60) (HSP-60) (Mitochondrial matrix protein P1) (HSP-65)
Seryl-tRNA synthetase (Serine--tRNA ligase) (SerRS) P26638 MOUSE Up (serin-tRNA ligase activity)
30 Accession Function Protein name Species Regulation # Stress-70 protein, mitochondrial precursor (75 kDa P38647 MOUSE Up glucose regulated protein) (GRP 75) (Peptide-binding protein 74) (PBP74) (P66 MOT) (Mortalin) (binding to p53)
26S proteasome non-ATPase regulatory subunit 13 Q9WVJ2 MOUSE Down (26S proteasome regulatory subunit S11) (26S proteasome regulatory subunit p40.5)
26S protease regulatory subunit 6B (MIP224) (MB67 P43686 HUMAN Down interacting protein) (TAT-binding protein-7) (TBP-7) (proteosome ATP binding)
S-adenosylmethionine synthetase alpha and beta forms Q00266 HUMAN Down (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III) (amino acid metabolism)
ATP synthase beta chain, mitochondrial precursor P56480 MOUSE Down (ion transporter activity, neucleotide binding)
Glutathione synthetase (Glutathione synthase) (GSH P51855 MOUSE Down synthetase) (GSH-S) (glutathion biosynthesis)
Methylcrotonyl-CoA carboxylase alpha chain, Q99MR8 MOUSE Down mitochondrial precursor (3-Methylcrotonyl-CoA carboxylase 1) (MCCase alpha subunit) (3- methylcrotonyl-CoA:carbon dioxide ligase alpha subunit) (biotin binding metabolism, ligase activity)
Lipid binding & Apolipoprotein E precursor (Apo-E) P08226 MOUSE Up transport Nonspecific lipid-transfer protein, mitochondrial P32020 MOUSE Up precursor (NSL-TP) (Sterol carrier protein 2) (SCP-2) (Sterol carrier protein X) (SCP-X) (SCPX) (protein peroxisome targeting, fatty acid binding, sterol carrier activity)
SEC14-like protein 2 (Alpha-tocopherol associated Q99J08 MOUSE Up protein) (TAP) (transcriptional activator activity, peptidase activity)
Microsomal triglyceride transfer protein, large O08601 MOUSE Down subunit precursor
31 Accession Function Protein name Species Regulation # Lipid binding & PCTP-like protein (PCTP-L) (StAR-related lipid Q9JMD3 MOUSE Down transport transfer protein 10) (StARD10) (START domain- containing protein 10) (Serologically defined colon cancer antigen 28 homolog) (may play specific roles in sperm maturation or fertilization) Hydrolase Proteasome subunit alpha type 4 (Proteasome Q9R1P0 MOUSE Up activity component C9) (Macropain subunit C9) (Multicatalytic endopeptidase complex subunit C9) (Proteasome subunit L) (endopeptidase activity, peptidase activity)
Proteasome subunit alpha type 5 (Proteasome zeta Q9Z2U1 MOUSE Up chain) (Macropain zeta chain) (Multicatalytic endopeptidase complex zeta chain) (endopeptidase activity, peptidase activity,)
Transitional endoplasmic reticulum ATPase (TER Q01853 MOUSE Up ATPase) (15S Mg(2+)-ATPase p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin] (ATP binding, cell growth or maintenace)
Calcium ion Calreticulin precursor (CRP55) (CALREGULIN) P14211 MOUSE Up binding (HACBP) (ERP60)
Transketolase (TK) (P68) P40142 MOUSE Down
Cell cycle gi|6754472|ref|NP_004847.2| kinesin family member 23 6754472 UNREAD Up regulation & isoform 2; mitotic kinesin-like 1; kinesin-like 5 (mitotic ABLE signal kinesin-like protein 1) [Homo sapiens] transduction Ribonuclease/angiogenin inhibitor 1 16307569 MUS Up MUSCUL US Zinc finger protein 189 O75820 HUMAN Down
RNA processing Heterogeneous nuclear ribonucleoproteins A2/B1 O88569 MOUSE Up (hnRNP A2 / hnRNP B1)
Poly(A) polymerase beta (PAP beta) (Polynucleotide Q9NRJ5 MOUSE Down adenylyltransferase beta) (Testis-specific poly(A) polymerase) (transferase activity)
Kinase ctivity Guanine nucleotide-binding protein beta subunit-like P25388 HUMAN Up protein 12.3 (P205) (Receptor of activated protein kinase C 1) (RACK1) (Receptor for activated C kinase)
Pyruvate kinase, isozymes R/L (L-PK) P53657 MOUSE Down
32 Accession Function Protein name Species Regulation # Transferase 10-formyltetrahydrofolate dehydrogenase (10- Q8R0Y6 MOUSE Down activity FTHFDH) (biosynthsis)
Thiosulfate sulfurtransferase (Rhodanese) P52196 MOUSE Down
Cytoskeleton Keratin, type I cytoskeletal 18 (Cytokeratin 18) P05784 MOUSE Down related (Cytokeratin endo B) (Keratin D)
Keratin, type II cytoskeletal 8 (Cytokeratin 8) P11679 MOUSE Down (Cytokeratin endo A)
Aflatoxin Aflatoxin B1 aldehyde reductase 1 27527042 MUS Up biosynthesis MUSCUL US Isomerase Triosephosphate isomerase (TIM) P17751 MOUSE Up activity (glycolysis, fatty acid biosynthesis)
Protein bio 60S acidic ribosomal protein P0 (L10E) P14869 MOUSE Down synthesis Cell mobility Actin-like protein 3 (Actin-related protein 3) (Actin-2) P32391 HUMAN Down
Lyase activity & Alpha enolase (2-phospho-D-glycerate hydro-lyase) P17182 MOUSE Down hydratase activity (Non-neural enolase) (NNE) (Enolase 1) (glycolysis)
GTP binding Elongation factor 2 (EF-2) P05197 RAT Down (translation elongation factor activity)
Aminoacylase gi|13384746|ref|NP_079647.1| aminoacylase 1 [Mus 13384746 UNREAD Down activity musculus] ABLE
Ligase activity Succinyl-CoA ligase[GDP-forming] beta-chain, Q9Z2I8 MOUSE Down mitochondrial precursor(Succinyl-CoA synthetase, betaG chain) (SCS-betaG) (GTP-specific succinyl-CoA synthetase beta subunit)
Not matched to Serine/threonine protein phosphatase 2A, 65 KDA P30153 HUMAN Down functional regulatory subunit A, alpha isoform (PP2A, subunit A, annotation PR65-alpha isoform) (PP2A, subunit A, R1-alpha isoform) (Medium tumor antigen-associated 61 KDA protein)
gi|31982147|ref|NP_775547.2|hexose-6-phosphate 31982147 UNREAD Down dehydrogenase (glucose 1-dehydrogenase) ABLE [Mus musculus] Selenium-binding protein 1 (56 KDa selenium-binding P17563 MOUSE Up protein) (SP56) Selenium-binding protein 2 (56 kDa acetaminophen- Q63836 MOUSE Up binding protein) (AP56)
33 Accession Function Protein name Species Regulation # Not matched to gi|13385656|ref|NP_080428.1| RIKEN cDNA 13385656 UNREAD Up functional 0610010D20 [Mus musculus] ABLE annotation gi|19527178|ref|NP_598721.1| RIKEN cDNA 19527178 UNREAD Up 9130231C15 [Mus musculus] ABLE
gi|21312204|ref|NP_077219.1| RIKEN cDNA 21312204 UNREAD Up 2810435D12 [Mus musculus] ABLE
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold] These results were categories by http://www.ebi.ac.uk/ego/
34 B. Identification of specifically expressed proteins related with the functional loss of p53 by 2-DE and MALDI-TOF MS
It was 44 proteins changed > 3-fold up-regulation or < 1/3-fold down-regulation in the liver of p53KO compared to B6 mice (Table 4). 44 proteins were found to be affected by p53 knock-out. 44 proteins search for protein function by http://www.ebi.ac.uk/ego/ (Table 5).
Among them, oxidoreductase activity (metabolism resulting in cell Growth) related proteins were 6. For example, aldehyde dehydrogenase 1A1, adehyde dehydrogenase mitochondrial precursor, glyceraldehyde 3-phosphate dehydrogenase, 2- oxoisovalerate dehydrogenase alpha subnuit mitochondrial precursor, short chain 3- hydroxyacyl-CoA dehydrogenase mitochondrial precursor were up-regulated proteins, dimethylglycine dehydrogenase was down-regulated protein. ATP binding and chaperone activity related proteins were 7. For example, ATP synthase alpha chain mitochondrial precursor, stress-70 protein mitochondrial precursor, transitional endoplasmic reticulum ATPase were up-regulated proteins. Heat shock protein 75kDa mitochondrial precursor, methylcrotonyl-CoA carboxylase alpha chain mitochondrial precursor, S-adenosylmethionine synthetase alpha and beta forms, T-complex protein
1 were down-regulated proteins. Lipid binding and transport related proteins were 5.
For example, apolipoprotein E precursor, fatty acid–binding protein, nonspecific lipid transfer protein mitochondrial precursor were up-regulated proteins. SEC14-like
35 protein 2, serum albumin precursor were down-regulated proteins. Down-regulated dihydropyrimidinase was hydrolase activity related protein. Calcium ion binding related proteins were 2. Calreticulin precursor and transketolase were down-regulated proteins. Cell cycle regulation and signal transduction related proteins were 2. They were 14-3-3 protein epsilon and ribosome-binding protein 1. All of them were down- regulated proteins. Up-regulated guanine nucleotide-binding protein beta subunit-like protein 12.3, adenosine kinase and down-regulated ketohexokinase were related with kinase activity. Up-regulated betaine-Homocysteine S-methyltransferase and Down- regulated 10-formyltetrahydrofolate dehydrogenase, ornithine carbamoyltransferase mitochondrial precursor, serine hydroxymethyltransferase, thiosulfate sulfurtransferase were related with transferase activity. There were 3 proteins have
Lyase activity and hydratase activity. Alpha enolase, probable urocanate hydratase and cystathionine beta-synthase were down-regulated proteins. Arginase activity related proteins were 3. There were arginase 1, hydrozymethylglutaryl-CoA lyase mitochondrial precursor and phosphoenolpyruvate carboxykinase. All of them were down-regulated proteins. Remaining 5 proteins were aflatoxin biosynthesis, isomerase activity, cell mobility, GTP binding aminoacylase activity related proteins.
Function of 2 proteins could not search.
36 Table 4. Lists of up and down-regulated proteins in the liver of p53KO mice compared to B6 mice
B6 vs p53KO
Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Actin-like protein 3 (Actin-related protein P32391 53 47372/5.6 HUMAN Up 3) (Actin-2) Aldehyde dehydrogenase 1A1 (Aldehyde P24549 36 54450/7.9 MOUSE Up dehydrogenase, cytosolic) (ALDH class 1) (ALHDII) (ALDH-E1)
Aldehyde dehydrogenase, mitochondrial P47738 30 56538/7.5 MOUSE Up precursor (ALDH class 2) (AHD-M1) (ALDHI) (ALDH-E2)
Apolipoprotein E precursor (Apo-E) P08226 24 35867/5.6 MOUSE Up ATP synthase alpha chain, mitochondrial Q03265 28 59753/9.2 MOUSE Up precursor Betaine—Homocysteine S- O35490 40 45021/8.0 MOUSE Up methyltransferase Fatty acid-binding protein, liver (L- P12710 54 14246/8.6 MOUSE Up FABP) (14 kDa selenium-binding protein)
gi|19527306|ref|NP_598840.1| adenosine 19527306 27 40149/5.8 UNREADABLE Up kinase [Mus musculus] M Glyceraldehyde 3-phosphate P16858 28 35810/8.4 MOUSE Up dehydrogenase (GAPDH) Guanine nucleotide-binding protein beta P25388 70 35077/7.6 HUMAN Up subunit-like protein 12.3 (P205) (Receptor of activated protein kinase C 1) (RACK1) (Receptor for activated C kinase)
Nonspecific lipid-transfer protein, P32020 23 59126/7.2 MOUSE Up mitochondrial precursor (NSL-TP) (Sterol carrier protein 2) (SCP-2) (Sterol carrier protein X) (SCP-X) (SCPX)
2-oxoisovalerate dehydrogenase alpha P11960 53 50165/7.7 RAT Up subunit, mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha) Short chain 3-hydroxyacyl-CoA Q61425 36 34464/8.8 MOUSE Up dehydrogenase, mitochondrial precursor (HCDH) (Medium and short chain L-3- hydroxyacyl-coenzyme A dehydrogenase)
37 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Stress-70 protein, mitochondrial P38647 27 73529/5.9 MOUSE Up precursor (75 kDa glucose regulated protein) (GRP 75) (Peptide-binding protein 74) (PBP74) (P66 MOT) (Mortalin) Transitional endoplasmic reticulum Q01853 36 89309/5.1 MOUSE Up ATPase (TER ATPase) (15S Mg(2+)- ATPase p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin]
Aflatoxin B1 aldehyde reductase 1 27527042 40 37677/6.4 MOUSE Down
Alpha enolase (2-phospho-D-glycerate P17182 23 47141/6.4 MOUSE Down hydro-lyase) (Non-neural enolase) (NNE) (Enolase 1) Arginase 1 (Liver-type arginase) Q61176 24 34808/6.5 MOUSE Down
Calreticulin precursor (CRP55) P14211 35 47995/4.3 MOUSE Down (CALREGULIN) (HACBP) (ERP60)
Cystathionine beta-synthase (Serine P32232 17 61455/6.1 RAT Down sulfhydrase) (Beta-thionase) (Hemoprotein H-450)
Dihydropyrimidinase (DHPase) Q9EQF5 46 56725/6.7 MOUSE Down (Hydantoinase) (DHP)
Dimethylglycine dehydrogenase, Q63342 17 96048/6.9 RAT Down mitochondrial precursor (ME2GLYDH)
Elongation factor 2 (EF-2) P05197 39 95285/6.4 RAT Down
10-formyltetrahydrofolate dehydrogenase Q8R0Y6 28 98710/5.6 MOUSE Down (10-FTHFDH)
gi|13384746|ref|NP_079647.1| 13384746 53 45795/5.8 UNREADABLE Down aminoacylase 1 [Mus musculus]
gi|20149748|ref|NP_619606.1| sarcosine 20149748 37 101683/6.3 UNREADABLE Down dehydrogenase [Mus musculus] M
Heat shock protein 75 kDa, mitochondrial Q9CQN1 35 80210/6.2 MOUSE Down precursor (HSP 75) (Tumor necrosis factor type 1 receptor associated protein) (TRAP-1) (TNFR-associated protein 1)
Hydroxymethylglutaryl-coA lyase, P38060 28 34161/8.6 MOUSE Down mitochondrial precursor (HMG-COA LYASE) (HL) (3-hydroxy-3- methylglutarate-coA lyase)
Ketohexokinase (Hepatic fructokinase) P97328 28 32751/5.8 MOUSE Down
38 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Methylcrotonyl-CoA carboxylase alpha Q99MR8 17 79345/7.7 MOUSE Down chain, mitochondrial precursor (3- Methylcrotonyl-CoA carboxylase 1) (MCCase alpha subunit) (3- methylcrotonyl-CoA:carbon dioxide ligase alpha subunit) Methylmalonyl-CoA mutase, P16332 32 82966/6.7 MOUSE Down mitochondrial precursor (MCM)
Ornithine carbamoyltransferase, P11725 20 39765/8.8 MOUSE Down mitochondrial precursor (OTCase) (Ornithine transcarbamylase)
Phosphoenolpyruvate carboxykinase, Q9Z2V4 28 69355/6.2 MOUSE Down cytosolic [GTP] (Phosphoenolpyruvate carboxylase) (PEPCK-C)
Probable urocanate hydratase Q8VC12 34 74591/7.3 MOUSE Down (Urocanase) (Imidazolonepropionate hydrolase) 14-3-3 protein epsilon (Mitochondrial P42655 27 29174/4.6 HUMAN Down import stimulation factor L subunit) (Protein kinase C inhibitor protein-1) (KCIP-1) (14-3-3E) Ribosome-binding protein 1 (Ribosome Q99PL5 26 172881/9.4 MOUSE Down receptor protein) (mRRp)
S-adenosylmethionine synthetase alpha Q00266 29 43648/5.9 HUMAN Down and beta forms (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III) SEC14-like protein 2 (Alpha-tocopherol Q99J08 41 46301/6.7 MOUSE Down associated protein) (TAP)
Serine hydroxymethyltransferase, P50431 23 52585/6.5 MOUSE Down cytosolic (Serine methylase) (Glycine hydroxymethyltransferase) (SHMT)
Serine/threonine protein phosphatase 2A, P30153 32 65224/5.0 HUMAN Down 65 KDA regulatory subunit A, alpha isoform (PP2A, subunit A, PR65-alpha isoform) (PP2A, subunit A, R1-alpha isoform) (Medium tumor antigen- associated 61 KDA protein) Serum albumin precursor P07724 45 68693/5.7 MOUSE Down
T-complex protein 1, zeta subunit (TCP- P80317 34 58005/6.6 MOUSE Down 1-zeta) (CCT-zeta) (CCT-zeta-1)
Thiosulfate sulfurtransferase (Rhodanese) P52196 47 33466/7.7 MOUSE Down
39 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Transketolase (TK) (P68) P40142 27 67631/7.2 MOUSE Down
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold]
40 Table 5. Functional annotation of differentially expressed proteins in the liver of p53KO mice compared to B6 mice
Accession Function Protein name Species Regulation # Oxidoreductase Aldehyde dehydrogenase 1A1 (Aldehyde P24549 MOUSE Up activity dehydrogenase, cytosolic) (ALDH class 1) (ALHDII) (metabolism (ALDH-E1) resulting in cell (aldehyde degydrogenase activity) growth) Aldehyde dehydrogenase, mitochondrial precursor P47738 MOUSE Up (ALDH class 2) (AHD-M1) (ALDHI) (ALDH-E2) (aldehyde degydrogenase activity)
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) P16858 MOUSE Up (glycolysis)
2-oxoisovalerate dehydrogenase alpha subunit, P11960 RAT Up mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha)
Short chain 3-hydroxyacyl-CoA dehydrogenase, Q61425 MOUSE Up mitochondrial precursor (HCDH) (Medium and short chain L-3-hydroxyacyl-coenzyme A dehydrogenase) (fatty acid metabolism)
Dimethylglycine dehydrogenase, mitochondrial Q63342 RAT Down precursor (ME2GLYDH) (electron transport)
ATP binding and ATP synthase alpha chain, mitochondrial precursor Q03265 MOUSE Up chaperone activity Stress-70 protein, mitochondrial precursor (75 kDa P38647 MOUSE Up glucose regulated protein) (GRP 75) (Peptide-binding protein 74) (PBP74) (P66 MOT) (Mortalin) (binding to p53)
Transitional endoplasmic reticulum ATPase (TER Q01853 MOUSE Up ATPase) (15S Mg(2+)-ATPase p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin] ( cell growth or maintenace)
Heat shock protein 75 kDa, mitochondrial precursor Q9CQN1 MOUSE Down (HSP 75) (Tumor necrosis factor type 1 receptor associated protein) (TRAP-1) (TNFR-associated protein 1)
41 Accession Function Protein name Species Regulation # Methylcrotonyl-CoA carboxylase alpha chain, Q99MR8 MOUSE Down mitochondrial precursor (3-Methylcrotonyl-CoA carboxylase 1) (MCCase alpha subunit) (3- methylcrotonyl-CoA:carbon dioxide ligase alpha subunit) (biotin binding metabolism, ligase activity)
S-adenosylmethionine synthetase alpha and beta forms Q00266 HUMAN Down (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III) (amino acid metabolism)
T-complex protein 1, zeta subunit (TCP-1-zeta) (CCT- P80317 MOUSE Down zeta) (CCT-zeta-1)
Lipid binding & Apolipoprotein E precursor (Apo-E) P08226 MOUSE Up transport Fatty acid-binding protein, liver (L-FABP) (14 kDa P12710 MOUSE Up selenium-binding protein)
Nonspecific lipid-transfer protein, mitochondrial P32020 MOUSE Up precursor (NSL-TP) (Sterol carrier protein 2) (SCP-2) (Sterol carrier protein X) (SCP-X) (SCPX) (protein peroxisome targeting, fatty acid binding, sterol carrier activity)
SEC14-like protein 2 (Alpha-tocopherol associated Q99J08 MOUSE Down protein) (TAP) (transcriptional activator activity, peptidase activity)
Serum albumin precursor P07724 MOUSE Down
Hydrolase Dihydropyrimidinase (DHPase) (Hydantoinase) (DHP) Q9EQF5 MOUSE Down activity
Calcium ion Calreticulin precursor (CRP55) (CALREGULIN) P14211 MOUSE Down binding (HACBP) (ERP60)
Transketolase (TK) (P68) P40142 MOUSE Down
Cell cycle 14-3-3 protein epsilon (Mitochondrial import P42655 HUMAN Down regulation & stimulation factor L subunit) (Protein kinase C signal inhibitor protein-1) (KCIP-1) (14-3-3E) transduction (protein folding, signal cascade)
Ribosome-binding protein 1 (Ribosome receptor Q99PL5 MOUSE Down protein) (mRRp)
42 Accession Function Protein name Species Regulation # Kinase activity Guanine nucleotide-binding protein beta subunit-like P25388 HUMAN Up protein 12.3 (P205) (Receptor of activated protein kinase C 1) (RACK1) (Receptor for activated C kinase) (signal transduction)
gi|19527306|ref|NP_598840.1| adenosine kinase [Mus 19527306 UNREAD Up musculus] M ABLE
Ketohexokinase (Hepatic fructokinase) P97328 MOUSE Down (ketohexokinase activity, transferase activity)
Transferase Betaine—Homocysteine S-methyltransferase O35490 MOUSE Up activity 10-formyltetrahydrofolate dehydrogenase (10- Q8R0Y6 MOUSE Down FTHFDH) (biosynthesis)
Ornithine carbamoyltransferase, mitochondrial P11725 MOUSE Down precursor (OTCase) (Ornithine transcarbamylase) (amino acid metabolism, arginine biosynthesis)
Serine hydroxymethyltransferase, cytosolic (Serine P50431 MOUSE Down methylase) (Glycine hydroxymethyltransferase) (SHMT)
Thiosulfate sulfurtransferase (Rhodanese) P52196 MOUSE Down
Aflatoxin aflatoxin B1 aldehyde reductase 1 27527042 MOUSE Down biosynthesis Isomerase Methylmalonyl-CoA mutase, mitochondrial precursor P16332 MOUSE Down activity (MCM)
Cell mobility Actin-like protein 3 (Actin-related protein 3) (Actin-2) P32391 HUMAN Up
Lyase activity & Alpha enolase (2-phospho-D-glycerate hydro-lyase) P17182 MOUSE Down hydratase (Non-neural enolase) (NNE) (Enolase 1) activity Probable urocanate hydratase (Urocanase) Q8VC12 MOUSE Down (Imidazolonepropionate hydrolase) (histidine, catabolism)
Cystathionine beta-synthase (Serine sulfhydrase) (Beta- P32232 RAT Down thionase) (Hemoprotein H-450) (amino acid metabolism, cystein biosynthesis)
Aminoacylase gi|13384746|ref|NP_079647.1| aminoacylase 1 [Mus 13384746 UNREAD Down activity musculus] ABLE
43 Accession Function Protein name Species Regulation # Arginase activity Arginase 1 (Liver-type arginase) Q61176 MOUSE Down
Hydroxymethylglutaryl-coA lyase, mitochondrial P38060 MOUSE Down precursor (HMG-COA LYASE) (HL) (3-hydroxy-3- methylglutarate-coA lyase) (catalytic activity)
Phosphoenolpyruvate carboxykinase, cytosolic [GTP] Q9Z2V4 MOUSE Down (Phosphoenolpyruvate carboxylase) (PEPCK-C) (lyase activity, lipid metabolism)
GTP binding Elongation factor 2 (EF-2) P05197 RAT Down (translation elongation factor activity)
Not matched to Serine/threonine protein phosphatase 2A, 65 KDA P30153 HUMAN Down functional regulatory subunit A, alpha isoform (PP2A, subunit A, annotation PR65-alpha isoform) (PP2A, subunit A, R1-alpha isoform) (Medium tumor antigen-associated 61 KDA protein)
gi|20149748|ref|NP_619606.1| sarcosine dehydrogenase 20149748 UNREAD Down [Mus musculus] M ABLE
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold] These results were categories by http://www.ebi.ac.uk/ego/
44 C. Identification of specifically expressed proteins in the liver HBxTg/p53KO double mutant mice by 2-DE and MALDI-TOF MS.
It was 31 proteins changed > 3-fold up-regulation or < 1/3-fold down-regulation in the liver of HBxTg/p53KO compared to B6 mice (Table 6). 31 proteins were found to be affected by HBx transgene and p53 knock-out. 31 proteins search for protein function by http://www.ebi.ac.uk/ego/ (Table 7).
Among them, Oxidoreductase activity (metabolism resulting in cell Growth) related proteins were 8. For example, acyl-CoA dehydrogenase long-chain specific mitochondrial precursor, acyl-CoA dehydrogenase medium-chain specific mitochondrial precursor, acyl-CoA dehydrogenase very long-chain specific mitochondrial precursor, aldehyde dehydrogenase 1A1, 2-oxoisovalerate dehydrogenase alpha subnuit mitochondrial precursor and phenylalanine-4- hydroxylase were up-regulated proteins. Dimethylglycine dehydrogenase and sulfite oxidase mitochondrial precursor were down-regulated proteins. ATP binding and chaperone activity related proteins were 4. For example, transitional endoplasmic reticulum ATPase is up-regulated protein. Heat shock protein 75KDa mitochondrial precursor, pyruvate carboxylase mitochondrial precursor and S-adenosylmethionine synthetase alpha and beta forms were down-regulated proteins. Transferase activity related proteins were 7. 4-aminobutyrate aminotransferase mitochondrial precursor, aspartate amonotransferase, hydrozymethylglutaryl-CoA synthase mitochondrial precursor and serine hydroxymethyltrasnferase were up-regulated proteins. 10-
45 formyltetrahydrofolate dehydrogenase, guanidinoacetate N-methyltransferase and thiosulfate sulfurtransferase were down-regulated proteins. There were 3 proteins have Lyase activity and hydratase activity. Lactoylflutathione lyase and probable urocanate hydratase were up-regulated proteins. Alpha enolase is down-regulated protein. Remaining 8 proteins were lipid binding and transport, hydrolase activity, calcium ion binding, aflatoxin biosynthesis, cell cycle regulation and signal transduction, ligase activity, malate metabolism and actin binding related proteins.
Function of 1 protein could not search.
46 Table 6. Lists of up and down-regulated proteins in the liver of HBxTg/p53KO double mutant mice compared to B6 mice
B6 vs HBxTg/p53KO
Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI 4-aminobutyrate aminotransferase, P50554 36 56530/9.0 RAT Up mitochondrial precursor (Gamma- Amino-N-Butyrate Transaminase) (GABA transaminase) (GABA aminotransferase) (GABA-AT) Acyl-CoA dehydrogenase, long-chain P51174 21 47908/8.5 MOUSE Up specific, mitochondrial precursor (LCAD)
Acyl-CoA dehydrogenase, medium-chain P45952 24 46482/8.6 MOUSE Up specific, mitochondrial precursor (MCAD) Acyl-CoA dehydrogenase, very-long-chain P50544 35 70876/8.9 MOUSE Up specific, mitochondrial precursor (VLCAD) (MVLCAD) Aldehyde dehydrogenase 1A1 (Aldehyde P24549 34 54450/7.9 MOUSE Up dehydrogenase, cytosolic) (ALDH class 1) (ALHDII) (ALDH-E1)
Aspartate aminotransferase, cytoplasmic P05201 49 46232/6.7 MOUSE Up (Transaminase A) (Glutamate oxaloacetate transaminase-1)
Calreticulin precursor (CRP55) P14211 40 47995/4.3 MOUSE Up (CALREGULIN) (HACBP) (ERP60)
Hydroxymethylglutaryl-CoA synthase, P54869 26 53787/8.0 MOUSE Up mitochondrial precursor (HMG-CoA synthase) (3-hydroxy-3-methylglutaryl coenzyme A synthase) Lactoylglutathione lyase Q9CPU0 53 20810/5.2 MOUSE Up (Methylglyoxalase) (Aldoketomutase) (Glyoxalase I) (Glx I) (Ketone-aldehyde mutase) (S-D-lactoylglutathione methylglyoxal lyase) NADP-dependent malic enzyme (NADP- P06801 38 63999/7.2 MOUSE Up ME) (Malic enzyme 1)
2-oxoisovalerate dehydrogenase alpha P11960 53 50165/7.7 RAT Up subunit, mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha)
47 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI Phenylalanine-4-hydroxylase (PAH) (Phe- P16331 43 51929/6.0 MOUSE Up 4-monooxygenase)
Probable urocanate hydratase Q8VC12 28 74591/7.3 MOUSE Up (Urocanase) (Imidazolonepropionate hydrolase) Serine hydroxymethyltransferase, P50431 44 52585/6.5 MOUSE Up cytosolic (Serine methylase) (Glycine hydroxymethyltransferase) (SHMT)
Transitional endoplasmic reticulum Q01853 29 89309/5.1 MOUSE Up ATPase (TER ATPase) (15S Mg(2+)- ATPase p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin] Aflatoxin B1 aldehyde reductase 1 27527042 40 37677/6.4 MOUSE Down
Alpha enolase (2-phospho-D-glycerate P17182 50 47141/6.4 MOUSE Down hydro-lyase) (Non-neural enolase) (NNE) (Enolase 1) D-lactate dehydrogenase 33585872 24 51848/6.2 MUS Down MUSCULUS Dimethylglycine dehydrogenase, Q63342 17 96048/6.9 RAT Down mitochondrial precursor (ME2GLYDH)
Eukaryotic translation initiation factor 3 Q9QZD9 20 36461/5.4 MOUSE Down subunit 2 (eIF-3 beta) (eIF3 p36) (eIF3i) (TGF-beta receptor interacting protein 1) (TRIP-1) 10-formyltetrahydrofolate dehydrogenase Q8R0Y6 45 98710/5.6 MOUSE Down (10-FTHFDH)
Fructose-1,6-bisphosphatase (D-fructose- Q9QXD6 31 36913/6.1 MOUSE Down 1,6-bisphosphate 1-phosphohydrolase) (FBPase) Glutamine synthetase (Glutamate-- P15105 33 42146/6.5 MOUSE Down ammonia ligase) Guanidinoacetate N-methyltransferase O35969 45 26336/5.4 MOUSE Down
Heat shock protein 75 kDa, mitochondrial Q9CQN1 35 80210/6.2 MOUSE Down precursor (HSP 75) (Tumor necrosis factor type 1 receptor associated protein) (TRAP-1) (TNFR-associated protein 1)
Pyruvate carboxylase, mitochondrial Q05920 28 129686/6.3 MOUSE Down precursor (Pyruvic carboxylase) (PCB)
48 Accession Sequence M.W Protein name Species Regulation # coverage(%) (Da)//pI S-adenosylmethionine synthetase alpha Q00266 29 43648/5.9 HUMAN Down and beta forms (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III) Serum albumin precursor P07724 35 68693/5.7 MOUSE Down
Sulfite oxidase, mitochondrial precursor Q8R086 19 54049/5.7 MOUSE Down
Thiosulfate sulfurtransferase (Rhodanese) P52196 47 33466/7.7 MOUSE Down
Vitamin D-binding protein precursor P21614 29 53086/5.3 MOUSE Down (DBP) (Group-specific component) (GC- globulin) (VDB)
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold]
49 Table 7. Functional annotation of differentially expressed proteins in the liver of HBxTg/p53KO double mutant mice compared to B6 mice
Accession Function Protein name Species Regulation # Oxidoreductase Acyl-CoA dehydrogenase, long-chain specific, P51174 MOUSE Up activity mitochondrial precursor (LCAD) (metabolism (acyl-coA dehydrogenase activity, electron transport, resulting in cell fatty acid metabolism) growth) Acyl-CoA dehydrogenase, medium-chain specific, P45952 MOUSE Up mitochondrial precursor (MCAD) (acyl-coA dehydrogenase activity, electron transport, fatty acid metabolism)
Acyl-CoA dehydrogenase, very-long-chain specific, P50544 MOUSE Up mitochondrial precursor (VLCAD) (MVLCAD) (acyl-coA dehydrogenase activity, electron transport, fatty acid metabolism)
Aldehyde dehydrogenase 1A1 (Aldehyde P24549 MOUSE Up dehydrogenase, cytosolic) (ALDH class 1) (ALHDII) (ALDH-E1) (aldehyde degydrogenase activity) 2-oxoisovalerate dehydrogenase alpha subunit, P11960 RAT Up mitochondrial precursor (Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain) (BCKDH E1-alpha)
Phenylalanine-4-hydroxylase (PAH) (Phe-4- P16331 MOUSE Up monooxygenase) (iron ion binding, amino acid binding) Dimethylglycine dehydrogenase, mitochondrial Q63342 RAT Down precursor (ME2GLYDH) (electron transport)
Sulfite oxidase, mitochondrial precursor Q8R086 MOUSE Down (electron transport)
ATP binding and Transitional endoplasmic reticulum ATPase (TER Q01853 MOUSE Up chaperone ATPase) (15S Mg(2+)-ATPase p97 subunit) (Valosin activity containing protein) (VCP) [Contains: Valosin] (cell growth or maintenace)
Heat shock protein 75 kDa, mitochondrial precursor Q9CQN1 MOUSE Down (HSP 75) (Tumor necrosis factor type 1 receptor associated protein) (TRAP-1) (TNFR-associated protein 1)
50 Accession Function Protein name Species Regulation # ATP binding and Pyruvate carboxylase, mitochondrial precursor Q05920 MOUSE Down chaperone (Pyruvic carboxylase) (PCB) activity (lipid biosynthesis, ligase activity)
S-adenosylmethionine synthetase alpha and beta forms Q00266 HUMAN Down (Methionine adenosyltransferase) (AdoMet synthetase) (MAT-I/III) (amino acid metabolism)
Lipid binding & Serum albumin precursor P07724 MOUSE Down transport
Hydrolase activity Fructose-1,6-bisphosphatase (D-fructose-1,6- Q9QXD6 MOUSE Down bisphosphate 1-phosphohydrolase) (FBPase)
Calcium ion Calreticulin precursor (CRP55) (CALREGULIN) P14211 MOUSE Up binding (HACBP) (ERP60)
Transferase 4-aminobutyrate aminotransferase, mitochondrial P50554 RAT Up activity precursor (Gamma-Amino-N-Butyrate Transaminase) (GABA transaminase) (GABA aminotransferase) (GABA-AT)
Aspartate aminotransferase, cytoplasmic P05201 MOUSE Up (Transaminase A) (Glutamate oxaloacetate transaminase-1) (amino acid metabolism, biosynthesis) Hydroxymethylglutaryl-CoA synthase, mitochondrial P54869 MOUSE Up precursor (HMG-CoA synthase) (3-hydroxy-3- methylglutaryl coenzyme A synthase) (acetyl coA metabolism, cholesterol biosynthesis)
Serine hydroxymethyltransferase, cytosolic (Serine P50431 MOUSE Up methylase) (Glycine hydroxymethyltransferase) (SHMT)
10-formyltetrahydrofolate dehydrogenase (10- Q8R0Y6 MOUSE Down FTHFDH) (biosynthesis) Guanidinoacetate N-methyltransferase O35969 MOUSE Down
Thiosulfate sulfurtransferase (Rhodanese) P52196 MOUSE Down
Aflatoxin Aflatoxin B1 aldehyde reductase 1 27527042 MOUSE Down biosynthesis
51 Accession Function Protein name Species Regulation # Cell cycle Eukaryotic translation initiation factor 3 subunit 2 Q9QZD9 MOUSE Down regulation & (eIF-3 beta) (eIF3 p36) (eIF3i) (TGF-beta receptor signal interacting protein 1) (TRIP-1) transduction
Lyase activity & Lactoylglutathione lyase (Methylglyoxalase) Q9CPU0 MOUSE Up hydratase activity (Aldoketomutase) (Glyoxalase I) (Glx I) (Ketone- aldehyde mutase) (S-D-lactoylglutathione methylglyoxal lyase) (carbohydrate metabolism)
Probable urocanate hydratase (Urocanase) Q8VC12 MOUSE Up (Imidazolonepropionate hydrolase) (histidine, catabolism)
Alpha enolase (2-phospho-D-glycerate hydro-lyase) P17182 MOUSE Down (Non-neural enolase) (NNE) (Enolase 1) (glycolysis)
Ligase activity Glutamine synthetase (Glutamate--ammonia ligase) P15105 MOUSE Down (nitrogen fixation)
Malate NADP-dependent malic enzyme (NADP-ME) (Malic P06801 MOUSE Up metabolism enzyme 1) (malate degydrogense activity – NAD dependent)
Actin binding Vitamin D-binding protein precursor (DBP) (Group- P21614 MOUSE Down specific component) (GC-globulin) (VDB)
Not matched to D-lactate dehydrogenase 33585872 MUS Down functional MUSCUL annotation US
Up-regulated : [> 3-fold], Down-regultaed : [< 1/3-fold] These results were categories by http://www.ebi.ac.uk/ego/
52 D. Summary of specifically expressed proteins profiles among groups.
After we identified up- and down- regulated proteins in each pair (Table 2, 4 and 6), all of the proteins profiles were compared with each other. Expressed proteins profiling were divided into three groups according to their expressed pattern in different groups (Table 8).
Group 1 proteins were differentially expressed proteins in the liver of both HBxTg mice and p53KO mice compared to B6 mice. These 12 proteins group included oxidoreductase activity (metabolism resulting in cell growth), lipid binding and transport, kinase activity, ATP binding and chaperone activity, GTP binding, calcium ion binding, aminoacylase activity and cell mobility related protein.
Group 2 proteins were differentially expressed proteins in the liver of both p53KO mice and HBxTg/p53KO mice compared to B6 mice. These 3 proteins were aldehyde dehydrogenase 1A1 related with oxidoreductase activity, probable urocanate hydratase related with lyase activity and hydratase activity, heat shock protein 75 kDa mitochondrial precursor (HSP 75) related with ATP binding and chaperone activity. 2-
D gel images of HSP 75 be shown in Fig 7A. This protein was dramatically down- regulated by 4-fold in pair 2 and by > 5-fold in pair 3.
Group 3 proteins were regulated proteins in all groups, HBxTg mice, p53KO mice and HBxTg/p53KO double mutant mice compared to B6 mice. 2-D gel images of group 3 proteins be shown total 9 proteins (Figs. 7B~J). 2-oxoisovalerate dehydrogenase alpha subunit related with oxidoreductase activity. This protein was
53 up-regulated by 3-fold in pair 1 and pair 2 and up-regulated by > 4-fold in pair 3 (Fig.
7B). Transitional endoplasmic reticulum ATPase related with ATP binding and chaperone activity. This protein was up-regulated by 3-fold in pair 1 and pair 2, and this protein up- regulated by 4-fold in pair 3 (Fig. 7C). S-adenosylmethionine synthetase alpha and beta forms were amino acid metabolism related protein. This protein was down-regulated by > 5-fold in pair 1 and by 4-fold in pair 2, but, down- regulated only by 3-fold in pair 3 (Fig. 7D). Alpha enolase (2-phospho-D-glycerate hydro-lyase) related with lyase activity and hydratase activity. This protein was down- regulated by 3-fold in pair 1 and pair 3. But, alpha enolase was down-regulated by >
4-fold in pair 2 (Fig. 7E). Dimethylglycine dehydrogenase mitochondrial precursor
(ME2GLYDH) related with oxidoreductase activity. This protein was down-regulated by > 5-fold in pair 1 and pair 2 but, ME2GLYDH was a little down-regulated in pair 3
(Fig 7F). 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) related with transferase activity. This protein was down-regulated by 4-fold in pair 1 and pair 2.
But, in pair 3, 10-FTHFDH disappeared almost (Fig. 7G). Thiosulfate sulfurtransferase (Rhodanese) related with transferase activity. This protein was down-regulated by 4-fold in pair 1 and pair 2. But, thiosulfate sulfurtransferase was little down-regulated in pair 3 (Fig. 7H). Aflatoxin B1 aldehyde reductase related with aflatoxin biosynthesis. This protein was up-regulated by 4-fold in pair 1, down- regulated by 3-fold in pair 2 and up-regulated by 5-fold in pair 3 (Fig. 7I).
Calreticulin precursor (CRP55) related with calcium ion binding. In particular, this protein dramatically changed in all pairs. This protein shifted their position as well as
54 changed their amount. This protein was up-regulated by > 4-fold in pair 1 and was down-regulated by 3-fold as well as changed one spot to two spots in pair 2.
Moreover, in pair 3, this protein was up-regulated a litter as well as changed one spots to three spots (Fig. 7J).
55 Table 8. Differentially expressed protein profiles of the liver in HBxTg, p53KO and HBxTg/p53KO double mutant mice compared to B6 mice
Regulation Protein name HBxTg/ Function HBxTg p53KO p53KO Group Aldehyde dehydrogenase, mitochondrial Oxidoreductase activity 1 precursor (ALDH class 2) (AHD-M1) (ALDHI) ↑ ↑ ㅡ (metabolism resulting in (ALDH-E2) cell growth) Lipid binding & Apolipoprotein E precursor (Apo-E) ↑ ↑ ㅡ transport
Guanine nucleotide-binding protein beta subunit-like protein 12.3 (P205) (Receptor of ↑ ↑ ㅡ Kinase activity activated protein kinase C 1) (RACK1) (Receptor for activated C kinase)
Nonspecific lipid-transfer protein, mitochondrial precursor (NSL-TP) (Sterol Lipid binding & ↑ ↑ ㅡ carrier protein 2) (SCP-2) (Sterol carrier transport protein X) (SCP-X) (SCPX)
Stress-70 protein, mitochondrial precursor (75 kDa glucose regulated protein) (GRP 75) ATP binding and ↑ ↑ ㅡ (Peptide-binding protein 74) (PBP74) (P66 chaperone activity MOT) (Mortalin)
Elongation factor 2 (EF-2) ↓↓ ↓↓ ㅡ GTP binding
Transketolase (TK) (P68) ↓↓ ↓↓ ㅡ Calcium ion binding
gi|13384746|ref|NP_079647.1| aminoacylase 1 ↓ ↓ ㅡ Aminoacylase activity [Mus musculus]
Methylcrotonyl-CoA carboxylase alpha chain, mitochondrial precursor (3-Methylcrotonyl- ATP binding and CoA carboxylase 1) (MCCase alpha subunit) ↓ ↓↓ ㅡ chaperone activity (3-methylcrotonyl-CoA:carbon dioxide ligase alpha subunit)
Serine/threonine protein phosphatase 2A, 65 KDA regulatory subunit A, alpha isoform Not matched to (PP2A, subunit A, PR65-alpha isoform) (PP2A, ↓ ↓↓ ㅡ functional annotation subunit A, R1-alpha isoform) (Medium tumor antigen-associated 61 KDA protein)
SEC14-like protein 2 (Alpha-tocopherol Lipid binding & ↑ ↓ ㅡ associated protein) (TAP) transport
Actin-like protein 3 (Actin-related protein 3) ↓ ↑ ㅡ Cell mobility (Actin-2)
56 Regulation Protein name HBxTg/ Function HBxTg p53KO p53KO Group 2 Aldehyde dehydrogenase 1A1 (Aldehyde Oxidoreductase activity dehydrogenase, cytosolic) (ALDH class 1) ↑ ↑ (metabolism resulting in (ALHDII) (ALDH-E1) cell growth)
Heat shock protein 75 kDa, mitochondrial precursor (HSP 75) (Tumor necrosis factor ATP binding and ↓↓ ↓↓↓ type 1 receptor associated protein) (TRAP-1) chaperone activity (TNFR-associated protein 1)
Probable urocanate hydratase (Urocanase) Lyase activity & ↓ ↑↑ (Imidazolonepropionate hydrolase) hydratase activity
Group 2-oxoisovalerate dehydrogenase alpha subunit, 3 Oxidoreductase activity mitochondrial precursor (Branched-chain ↑ ↑ ↑↑ (metabolism resulting in alpha-keto acid dehydrogenase E1 component cell growth) alpha chain) (BCKDH E1-alpha)
Transitional endoplasmic reticulum ATPase (TER ATPase) (15S Mg(2+)-ATPase p97 ATP binding and ↑ ↑ ↑↑ subunit) (Valosin containing protein) (VCP) chaperone activity [Contains: Valosin]
S-adenosylmethionine synthetase alpha and ATP binding and beta forms (Methionine adenosyltransferase) ↓↓↓ ↓↓ ↓ chaperone activity (AdoMet synthetase) (MAT-I/III)
Alpha enolase (2-phospho-D-glycerate hydro- Lyase activity & ↓ ↓↓ ↓ lyase) (Non-neural enolase) (NNE) (Enolase 1) hydratase activity
Oxidoreductase activity Dimethylglycine dehydrogenase, mitochondrial ↓↓↓ ↓↓ ↓ (metabolism resulting in precursor (ME2GLYDH) cell growth)
10-formyltetrahydrofolate dehydrogenase (10- ↓↓ ↓↓ ↓↓↓ Transferase activity FTHFDH)
Thiosulfate sulfurtransferase (Rhodanese) ↓↓ ↓↓ ↓ Sulfate transport
Aflatoxin B1 aldehyde reductase 1 ↑↑ ↓ ↓↓↓ Aflatoxin biosynthesis
Calreticulin precursor (CRP55) ↑↑ ↓ ↑ Calcium ion binding (CALREGULIN) (HACBP) (ERP60)
* ↓ : 3-fold change, ↓↓ : 4-fold change, ↓↓↓ : > 5-fold change
57
HBxTg/p53KO double B6 mice HBxTg mice p53KO mice mutant mice
A
B
C
D
E
58
HBxTg/p53KO double B6 mice HBxTg mice p53KO mice mutant mice
F
G
H
I
J
59 Figure 7. 2-DE images of differentially expressed proteins in the liver of B6,
HBxTg, p53KO and HBxTg/p53KO double mutant mice.
A. Heat shock protein 75 kDa, mitochondrial precursor (HSP 75) (Tumor necrosis
factor type 1 receptor associated protein) (TRAP-1) (TNFR-associated protein 1)
B. 2-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor
(Branched-chain alpha-keto acid dehydrogenase E1 component alpha chain)
(BCKDH E1-alpha)
C. Transitional endoplasmic reticulum ATPase (TER ATPase) (15S Mg(2+)-ATPase
p97 subunit) (Valosin containing protein) (VCP) [Contains: Valosin]
D. S-adenosylmethionine synthetase alpha and beta forms (Methionine
adenosyltransferase) (AdoMet synthetase) (MAT-I/III)
E. Alpha enolase (2-phospho-D-glycerate hydro-lyase) (Non-neural enolase) (NNE)
(Enolase 1)
F. Dimethylglycine dehydrogenase, mitochondrial precursor (ME2GLYDH)
G. 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH)
H. Thiosulfate sulfurtransferase (Rhodanese)
I. Aflatoxin B1 aldehyde reductase 1
J. Calreticulin precursor (CRP55) (Calregulin) (HACBP) (ERP60)
60 V. Discussion
We aimed to elucidate the molecular mechanism of HCC and to discover the effect of functional loss of p53 in the early HCC by HBx. In human, HCC incidence is consistently higher in man than in woman. This result has been reported to be similar in other transgenic mice which developed HCC. 9,29The liver weight in HBxTg, p53KO and HBxTg/p53KO double mutant mice were significantly increased compared to B6 mice. ALT level of HBxTg/p53KO double mutant mice was significantly higher than that of B6, HBxTg and p53KO mice. In previous study (not published), HBxTg/p53KO double mutant mice, HCC was developed in 5 month old
HBxTg/p53KO double mutant mice, however, HCC was formed in 9 month old
HBxTg mice. The development of HCC was accerelated in HBxTg/p53KO double mutant mice compared to HBxTg mice. Therefore, we indentified HBxTg/p53KO double mutant mice in 1 month old, when it was clinically normal, but genetically affected by HBx and p53.
We identified several proteins, which were differentially expressed in each pair.
We found cell cycle and several metabolism related protein in pair 1 (B6 vs HBxTg).
Also signal transduction pathway and ATP binding and chaperone activity related proteins were found. These proteins have function of cellular signal pathway. In previous reports, these protein profiles of cancers were deduced using 2-DE and
MALDI-TOF MS, and from these informations, cancer classifications, the
61 establishments of diagnostic madders, and selection of therapeutic target candidateds were sought 30,31,32. There were no examples of HCC protein profiles obtained by 2-
DE and MALDI-TOF MS the proteome changes have not been studied from
HBxTg/p53KO double mutant mice liver tissue. Therefore, in this study, 2-DE and
MALDI-TOF MS were employed to obtain the protein profile of HCC together with gain of HBx function and loss of p53 function. We analyzed proteome in mouse liver tissue when oncogenic HBx expressed and tumor suppress p53 repressed by using
HBxTg/p53KO mice
Comparing the liver with each other, a significant change in expression level was found in 24 proteins. Some of the representative proteins annotated in 2D were proteins related with lipid metabolism, fatty acid metabolism, cell cycle proteins and
ATP binding and chaperone activity.
In particular calreticulin precursor (CRP55) was considered HBx and p53 related protein in HCC. Calreticulin precursor (CRP55) was calcium ion binding related protein. This protein dramatically changed in all pairs. This protein shifted their position as well as changed their amount. This protein was considered amount of this protein was regulated by gain of HBx function and position of protein was shifted by loss of p53 function. In conclusion, the identified proteins, both up-regulated and down-regulated, may participate in the progression of HCC caused by the HBx with the functional loss of p53.
62 VI. Conclusion
To elucidate factors involved in HCC caused by HBx under the functional loss of p53, we employed two dimensional polyacrylamide gel electrophoresis (2DE) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-
TOF MS) and determined profiles of differentially expressed proteins in the liver of
1-month-old male HBxTg, p53KO and HBxTg/p53KO mice compared to its age- matched normal mice (B6).
Cell cycle, cytoskeleton and several metabolisms related proteins were mainly differentially expressed in the liver of HBxTg mice compared to B6 mice. Signal transduction pathway, ATP binding and chaperone activity and lipid binding related proteins were differentially expressed in the liver of p53KO mice compared to B6 mice. Cell cycle, several metabolisms, ATP binding and chaperone activity, transferase activity related proteins were differentially expressed in the liver of
HBxTg/p53KO double mutant mice compared to B6 mice.
We identified 24 liver proteins, which were differentially regulated in each of
HBxTg, p53KO and HBxTg/p53KO double mutant mice compared with B6 mice.
They were proteins related with lipid metabolism, fatty acid metabolism, ATP synthesis and several metabolism related proteins. Some of identified proteins,
Calreticulin precursor and Aflatoxin B1 aldehyde reductase 1 were significantly differentially regulated in the HBxTg mice under the functional loss of p53 compared
63 to B6 mice These proteins may be involved in the acceleration of HCC in
HBxTg/p53KO double mutant mice.
Our results may provide useful information for the understanding of molecular pathogenesis of HCC by HBx related with the functional loss of p53.
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69 Abstract in Korea
HBxTg/p53KO 이중형질전환 마우스의 간에서
프로테오믹스를 이용한 단백질 발현 양상 분석
박 은 숙
연세대학교 대학원 의과학과
<지도교수 안 용 호>
B 형간염 바이러스는 간세포 암의 병인 요소로 알려져 있다. B 형간염 바
이러스는 잠재적으로 발암 단백질인 HBx 를 암호화 하고 있다. 그러나 간
세포 암 발생에 있어서의 정확한 기전은 아직까지 잘 알려져 있지 않다.
다만 HBX 가 p53 유전자의 발현을 억제 또는 다른 유전자와 결합하는 것
을 억제함으로써 간세포의 증식을 유도하고 이러한 비정상적인 세포의 증
식이 간세포 암을 유발하는 것으로 알려져 있다. 이와 반대로 HBx 가 간
70 세포의 세포사멸을 유도한다는 보고도 있으나 그 과정에서 p53 의 관련성
에 대해서는 아직까지 불확실한 실정이다. 최근까지 많은 연구들이 있지만
HBx 에 의한 간세포 암 발생에 있어서 p53 의 기능에 대해서는 아직까지
불확실한 실정이다. 이에 본 연구에서는 HBx 에 의한 간세포 암의 발생에
관여하는 단백질들을 탐색하고 p53 유전자의 기능적 상실이 어떠한 영향을
미치는지 확인하기 위해 다음과 같은 실험을 하였다. HBx 형질전환 마우스
와 p53 knock-out 마우스를 교배하여 HBxTg/p53KO 이중형질전환 마우스를
생산한 후, 간 기능 검사를 수행하고 H & E 염색을 통해 간의 조직변화를
관찰하였다. HBxTg/p53KO 이중형질전환 마우스에서는 ALT 수치가 B6 마우
스, HBx 형질전환 마우스, p53 knock-out 마우스와 비교해 현저하게 높았다.
또한 HBxTg/p53KO 이중형질전환 마우스는 간세포의 팽창과 불규칙적인 hepatic cord 를 보였다. 2-DE 와 MALDI-TOF MS 를 이용하여 HBx 형질전
환 마우스, p53 knock-out 마우스, HBxTg/p53KO 이중형질전환 마우스의 1개
월령 간에서 다르게 발현되는 단백질을 분석하였다. 단백질 발현 분석결과
HBx 형질전환 마우스와 p53 knock-out 마우스, HBxTg/p53KO 이중형질전환
마우스 간 조직에서 정상 마우스인 B6 마우스 간 조직과 비교해 다르게
발현하는 단백질을 발견했고 중요하게 발현이 변화하는 24 개의 단백질을
발견했다. 이러한 단백질 중에는 cell cycle regulation, lipid metabolism, fatty acid metabolism, ATP synthesis 등과 관련된 단백질들이었다. 이러한 단백질들
71 의 발현 변화는 HBx 에 의한 간암발생이 p53 의 기능적 손실에 의해서 가
속되는데 관련이 있는 것으로 사료된다.
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핵심되는 말 : HBx, p53, HBxTg/p53KO 형질전환마우스, 간
72